The present invention relates to a method for detecting or identifying a base pair, which can not construct a normal complementary base pairing, in base pairs, which can not construct a normal complementary base pairing, comprising making said base pair which can not construct a normal base pairing construct a pseudo-base-pairing, and assaying a formation of said pseudo-base-pairing, a reagent therefor, a kit containing the same, a compound therefore, and a method for detecting an abnormal base sequence in DNA or RNA using said method.
In a construction of a double-stranded structure by a hybridization of nucleic acids such as DNA and RNA, a base pair for constructing a base pairing is fixed. For example, these combinations are guanine (G) and cytosine (C), and adenine (A) and thymine (T). Generally, all bases hybridize by constructing such base parings, but sometimes, there is a case not constructing such a base pairing in some part of a base sequence.
For example, when some DNA and another DNA are placed under a condition to hybridize, almost all bases can construct such base pairings, but there is a case that one or several bases, may not able to construct such a base pairing. In the present specification, such a base pair, which can not construct a normal base pairing, is designated as mismatched base pair.
Recently, studies on various hereditary diseases caused by differences in one or more bases are in progress. For example, a hereditary disease having a gene, in which one base is different from the normal one [SNP (Single Nucleotide Polymorphism)] is known, and an elucidation for the hereditary disease is attracting an attention. If such gene hybridize with a normal gene, most of bases can hybridize and construct normal base pairings, but a mismatched base pairing may occur in one base pair.
At present, as a method for detecting such a mismatched base pair, a mean for comparing a hybridization efficiency of a double-stranded DNA is generally known. However, since the method needs a great effort because base sequences of the DNA containing the mismatched base pair should be known in advance in order to use the method, this is not a suitable method for treating a large number of specimens. Further, another method utilizing a selective binding of a DNA repair protein such as MutS to an injured position of a gene is known, but it is difficult to maintain an activity of the protein.
As explained above, detection of the part of a mismatched base pair in a hybridized DNA is very difficult and sensitivity is very low, consequently an establishment of a simple and highly sensitive detection method has been requested.
The present inventors developed a bulge DNA recognition molecule, which was a molecule specifically binding to a DNA (bulge DNA) having an unpaired base (bulge base) generated in a double-stranded DNA and stabilizing the same (JP-A-11-262205). The bulge DNA recognition molecule not only forms a hydrogen bond with an unpaired base but also is stabilized by intercalating in a space formed by a presence of the bulge base by utilizing stacking interactions between an aromatic ring and bases near by the bulge.
The present inventors further studied an action to the unpaired base utilizing a stacking effect in the presence of surrounding bases, and found that even in a position at the mismatched base pair, a compound having two molecular species which could construct base pairings was incorporated into the space relatively stably by the stacking effect.
The present invention provides a simple and high sensitive method for detecting such a mismatched base pair.
More particularly, the present invention provides a simple and high sensitive method for detecting a mismatched base pair in a double-stranded DNA and a reagent therefor.
The present invention relates to a method for detecting or identifying a base pair, which can not construct a normal complementary base pairing, in base pairs, which can not construct a normal complementary base pairing, comprising using a compound represented by the general formula (I):
Axe2x80x94Lxe2x80x94Bxe2x80x83xe2x80x83(I)
(wherein A is a chemical structural moiety constructing a complementary base pairing with a base in the base pair which can not construct a normal base pairing, B is a chemical structural moiety constructing another complementary base pairing with the other base in the base pair which can not construct a normal base pairing, and L is a linker structure linking the chemical structural moieties A and B), having a chemical structural moiety A and a chemical structural moiety B, both of which can construct base pairings with corresponding bases of the base pair, respectively, and a linker structure L linking said chemical structural moieties A and B, constructing a pseudo-base-pairing to said base pair which can not construct a normal base pairing, and assaying the construction of said pseudo-base-pairing.
Further, the present invention relates to a reagent for constructing a pseudo-base-pairing with a base pair which can not construct a normal base pairing comprising a compound represented by the general formula (I):
Axe2x80x94Lxe2x80x94Bxe2x80x83xe2x80x83(I)
(wherein A is a chemical structural moiety constructing a complementary base pairing with a base of a base pair which can not construct a normal base pairing, B is a chemical structural moiety constructing another complementary base pairing with the other base of the base pair which can not construct a normal base pairing, and L is a linker structure linking with the chemical structural moieties A and B), for constructing pseudo-base-pairings with a base pair which can not construct a normal base pair, in the method described above for detecting or identifying a base pair, which can not construct a normal base pairing, by constructing pseudo-base-pairings with a base pair which can not construct a normal base pairing and assaying the construction of said pseudo-base-pair.
Still further, the present invention relates to a kit comprising consisting of the above described reagent of the present invention and materials for detecting or identifying a base pair, which can not construct a normal complimentary base pairing, by constructing pseudo-base-pairings in a base pair, which can not construct a normal base pairing, and assaying said pseudo-base-pairing.
Furthermore, the present invention relates to a compound represented by the general formula (II): 
(wherein R and R1 are a hydrogen atom, an alkyl group having 1 to 15 carbon atoms wherein one or more carbon atoms in said alkyl group are optionally substituted by oxygen atom or nitrogen atom, an alkoxy group having 1 to 15 carbon atoms wherein one or more carbon atoms in said alkoxy group are optionally substituted by oxygen atom or nitrogen atom, or a mono- or dialkylamino group having 1 to 15 carbon atoms wherein one or more carbon atoms in said alkylamino group are optionally substituted by oxygen atom or nitrogen atom; R2 is an alkyl group having 1 to 20 carbon atoms wherein one or more carbon atoms in said alkyl group are optionally substituted by oxygen atom, nitrogen atom or carbonyl group), or immobilized form of said compound modified to a chemical structure which enables to immobilize the compound on a plate or a detecting apparatus for an instrumental analysis.
Still furthermore, the present invention relates to a method for detecting an abnormal base sequence in DNA or RNA comprising hybridizing a single-stranded DNA or RNA of a specimen and a corresponding DNA or RNA thereto having a normal base sequence, subsequently constructing pseudo-base-pairings with a base pair, which can not construct a normal base pairing, in said hybridized DNA or RNA by using a compound represented by the general formula (I):
Axe2x80x94Lxe2x80x94Bxe2x80x83xe2x80x83(I)
(wherein A is a chemical structural moiety constructing a complementary base pairing with a base of a base pair which can not construct a normal base pairing, B is a chemical structural moiety constructing another complementary base pairing with the other base of the base pair which can not construct a normal base pairing, and L is a linker structure linking the chemical structural moieties A and B), having a chemical structural moiety A and a chemical structural moiety B, both of which can construct base pairings with corresponding bases of the base pair, respectively, and a linker structure L linking said chemical structural moieties A and B, assaying said pseudo-base-pairing, and detecting and identifying a base pair which can not construct a normal base pairing.
In the following explanation, the above description xe2x80x9ca chemical structural moiety constructing a complementary base pairing with a base in a base pair which can not construct a normal base pairing [the parts of A and B in the general formula (I)] is sometimes simply designated as xe2x80x9cbase recognition sitexe2x80x9d.